A method of optically measuring a sample which is called STED microscopy is known from U.S. Pat. No. 7,253,893 B2. To the end of fluorescence microscopically examining a sample, a fluorescent dye or fluorophore in the sample is first transferred into an excited energetic state by means of excitation light. In this optical excitation, the usual limit for spatial resolution in optical methods of λ/2n applies, λ being the wavelength of the light used and n being the diffraction index of the sample. To get below this limit, the optically excited state of the fluorophore is de-excited with de-excitation light except of a desired measuring point in which the intensity distribution of the de-excitation light has a zero point; i.e. the fluorophore is forced to stimulated emission everywhere outside the actual measuring point by means of the de-excitation light. The dimensions of the resulting fluorescent measuring point, i.e. the spatial resolution of the remaining fluorescence, can significantly be lowered below the usual optical resolution limit in that the de-excitation light is applied to the sample outside of the desired measuring point at such a high intensity that saturation is achieved in de-excitation by means of stimulated emission. Thus, the fluorophore in the sample remains in its fluorescent state in a very narrowly delimited area about the zero point of the intensity distribution of the de-excitation light only. To cope with a limited average intensity of the de-excitation light, the de-excitation light and also the excitation light are pulsed. A further reason for using pulsed light in STED microscopy is to avoid that the intensive de-excitation light stresses the sample, even if the dye is not excited. Regarding a same area of the sample, irradiation with both the excitation and the de-excitation light is repeated multiple times at a short repetition interval which is sufficiently above the half-life of the fluorescent state of the fluorophore of typically 1 ns and has an order of magnitude of 10 ns to have a measurement signal sufficiently standing out of the background noise even with only few fluorophore molecules within a measuring point. Pulses of the excitation light and of the de-excitation light incident in the same areas of the sample comprise a typical repetition rate of 80 MHz.
With a high intensity of the de-excitation light which is necessary for saturation of the de-excitation outside the actual measuring point, there is a considerable probability that the dye in the sample bleaches, i.e. that it is chemically changed in such a way that it does no longer emit fluorescence light. Thus, the lifetime of the dye, i.e. the number of times at which fluorescence light from it may be registered from the fluorophore, is considerably reduced. This delimits the yield of fluorescence light from an actual sample in which naturally only a limited number of fluorophore molecules is available.
Similar problems with regard to the yield of photons in optically measuring a sample also occur with other fluorescence microscopic methods working with pulsed light, i.e. if high light intensities are employed, such as in multi-photon excitation, and also in other methods of optically measuring a sample, like for example in the lifetime measurement of fluorophores (life-time-imaging).
To enhance the yield of registered photons in methods of optically measuring a sample, high efforts have been spent to enhance the responsiveness of detectors to incident photons by which the photons from a sample are registered. An increase in responsiveness of high value detectors from about 20% up to 40 to 60% has been achieved within the previous 10 to 15 years. The associated increase of the yield of registered photons by a maximum factor of 3 was, however, accompanied by an extreme increase in cost of these detectors.
Despite the improvements with regard to the responsiveness of the detectors used, it is still the limited total yield of photons emitted by the fluorophore which sets the limits to most fluorescence-based measuring methods. In fluorescence microscopy, the limited number of photons emitted by a fluorophore in total, i.e. up to bleaching, nearly always is the main problem. Each considerable increase in the absolute signal by means of increasing the number of fluorescence light emissions prior to bleaching is of general importance for fluorescence microscopy.
A further method of optically measuring a sample which is called confocal two-photon microscopy is known from U.S. Pat. 2002/0027202 A1. In two-photon microscopy a quadratic dependency of the transition probability on the intensity distribution of the excitation light, which the fluorophore displays with regard to a transition out of its ground state into its excited fluorescent state upon taking up energy of two photons, is used for resolution enhancement. The excitation light is concentrated to pulses of high intensity to obtain an as high as possible yield of fluorescent light here. Due to the quadratic dependency of the transition probability of the fluorescence dye on the intensity of the excitation light, these pulses of high intensity result in more fluorescence light with a same average power of the excitation light as compared to a higher number of pulses of lower intensity. U.S. Pat. 2002/0027202 A1 additionally considers a negative effect on the fluorescence light yield which may result from a saturation of the excitation of the fluorescent state during each single pulse, and proposes to adjust the intensity of the pulses by means of keeping their repetition rate at a constant power of the excitation light so high that the negative influence on the fluorescence light yield does just not yet occur. The danger of bleaching of the fluorescence dye is not considered. As principally possible with regard to the repetition rate of the excitation light pulses U.S. Pat. 2002/0027202 A1 indicates a range of kHz to GHz, the repetition rate being to be adapted to the lifetime of the fluorescence dye; no numerical example, however, being given for this adaptation.
From EP 0666 473 B1 it is known to achieve a comparatively high yield of fluorescence light in two-photon microscopy despite the use of excitation light of comparatively low power in that very special fluorescence dyes, like for example lanthanide chelates, which have a long average lifetime of at least 0.1 μs (1×10−7 s), are subjected to comparatively long pulses of excitation light adapted to these lifetimes. The repetition rate of these long pulses is lower than 10 MHz (1×107 Hz) which corresponds to the indicated lifetime in the usual way.
The lifetime of the fluorescent state of fluorescence dyes usually used in fluorescence microscopy, however, is in the order of magnitude of 1 ns, i.e. shorter than 10 ns.
There is the need of a method of optically measuring a sample by which a considerable increase in the yield of registered photons can be achieved without a further expensive increase of the responsiveness of the detectors used.